Method for synthesizing conformationally constrained peptides, peptidomimetics and the use thereof as synthetic vaccines

ABSTRACT

The present invention relates to methods for synthesizing conformationally constrained peptides and cyclic peptidomimetics obtainable by these methods which are conformationally constrained due an internal cross-link. This cross-link is formed between the side chain of an amino acid residue or analog and a (2S, 4S)4-functionalized proline residue. The invention further relates to the use of (2S, 4S)-4-functionalized proline residues as building units in the synthesis of such peptidomimetics and to the use thereof as antigens, alone or in combination with suitable immunopotentiating delivery systems, for example immunopotentiating reconstituted influenza virosomes to elicit an immune response in a mammal. Moreover, the invention also relates to pharmaceutical compositions containing the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. Ser. No. 10/559,010, filed Mar. 20, 2006,which is a National Stage Entry of PCT/EP04/05952, filed Jun. 2, 2004and which claims foreign priority to EP 03012520.7 filed Jun. 2, 2003.The disclosures of each of these applications are incorporated herein byreference in their entirety.

DESCRIPTION

The present invention relates to methods for synthesizingconformationally constrained peptides and cyclic peptidomimeticsobtainable by these methods which are conformationally constrained duean internal cross-link. This cross-link is formed between the side chainof an amino acid residue or analog and a (2S,4S)-4-functionalizedproline residue. The invention further relates to the use of(2S,4S)-4-functionalized proline residues as building units in thesynthesis of such peptidomimetics and to the use thereof as antigens,alone or in combination with suitable immunopotentiating deliverysystems, for example immunopotentiating reconstituted influenzavirosomes to elicit an immune response in a mammal. Moreover, theinvention also relates to compositions containing the same.

New scientific knowledge and technologies allow to identify and localizeessential neutralizing epitopes of microorganisms causing infections andother diseases. These antigen structures can often be mimicked usingsmall linear peptides or polypeptides which are generally designated aspeptidomimetics. Such mimetics appear to be promising candidates forvaccination strategies and in the design of novel synthetic vaccines.

However, conformation plays a key role in the ability of peptides toelicit antibody responses against folded proteins. Linear peptides oftenelicit antibodies that bind well to denatured proteins, but lessfrequently recognize conformational epitopes in native proteinstructures. In general, these peptides are more conformationally mobileand therefore unlikely to adopt a stable secondary structure in aqueoussolution which corresponds to the three-dimensional epitope on thesurface of a protein.

To overcome these limitations, conformational constraints can beincorporated into the primary peptide chain which will reduce the degreeof structural flexibility. If the “frozen” conformation of such apeptide imitates the corresponding secondary structure of the targetepitope, the peptide antigen can be used to raise antibodies whichpotentially cross-react with protein structures bearing such epitopes.

There are several approaches known in the prior art to introduceconformational constraints into a linear peptide or polypeptide chain.For example, bridging between two neighbouring amino acids in a peptideleads to a local conformational modification, the flexibility of whichis limited in comparison with that of regular peptides. Somepossibilities for forming such bridges include incorporation of lactamsand piperazinones (for review see Giannis & Kolter, Angew. Chem. Int.Ed., 1993, 32: 1244).

Global restrictions in the conformation of a peptide are possible bylimiting the flexibility of the peptide strand through cyclization(Hruby et al., Biochem. J., 268:249, 1990). The common modes ofcyclization are the same found in naturally occurring cyclic peptides.These include side chain to side chain cyclization or side chain toend-group cyclization. Another common cyclization is the end-to-endcyclization.

Another conceptual approach to the conformational constraint of peptideswas introduced by Gilon, et al., (Biopolymers, 31:745, 1991) whoproposed backbone to backbone cyclization of peptides. The theoreticaladvantages of this strategy include the ability to effect cyclizationvia the carbons or nitrogens of the peptide backbone without interferingwith side chains that may be crucial for interaction with the antibody.

Yet another approach in the design of conformationally constrainedpeptidomimetics, which is described in U.S. Ser. No. 10/114,918, is toattach a short amino acid sequence of interest to a template, togenerate a cyclic constrained peptidomimetic. Such cyclicpeptidomimetics are not only structurally stabilized by their templates,and thereby offer three-dimensional conformations that may imitateconformational epitopes on viruses and parasites, but they are also moreresistant than linear peptides to proteolytic degradation in serum.

U.S. Ser. No. 10/114,918 further discloses the synthesis ofconformationally constrained cross-linked peptidomimetics by preparationof synthetic amino acids for backbone coupling to appropriatelypositioned amino acids in order to stabilize the supersecondarystructure of, mimetics. Cross-linking can be achieved by amide couplingof the primary amino group of an orthogonally protected(2S,3R)-3-aminoproline residue to a suitably positioned side chaincarboxyl group of glutamate. This approach has been followed in thepreparation of conformationally constraint tetrapeptide repeats of theCS protein wherein at least one proline has been replaced by2S,3R)-3-aminoproline and, in order to introduce a side chain carboxylgroup, glutamate has been incorporated as a replacement for alanine.

There are several approaches in the prior art to use modified prolineanalogs for incorporating conformational constraints into peptides. Forexample, Zhang et al., J. Med. Chem., 1996, 39: 2738-2744, describessynthesis and biological testing of several cyclic analogs ofangiotensin II being cyclized between positions 5 and 7 to study theconformational features of molecular recognition of angiotensin II.Cyclization has been achieved by forming amide bonds between4-amino-trans-proline and side chain carboxyl groups of aspartate andglutamate on one hand and on the other hand, by disulfide bridgesbetween homocysteine residues and 4-mercapto-trans/cis-prolines.

Furthermore, (2S,3R,4R)-diaminoproline as a diketopiperazine wasincorporated by solid-phase peptide synthesis into a protein loopmimetic. This was shown by NMR analysis to adopt a stable beta-hairpinconformation in DMSO (Pfeifer & Robinson, Chem. Comm., 1998, 1977-1978).

Vaccination is arguably the most successful medical invention and globalvaccination programs have yielded impressive results. However, theappearance of novel infectious diseases of global threat, like AIDS andthe comeback of infectious diseases that seemed to be mastered bychemotherapy, like tuberculosis and malaria causes a great need todevelop new sophisticated vaccines.

In particular, malaria is an increasing health problem throughout thethird world. Several hundred million people suffer from the disease andthe most acute form, caused by the protozoan parasite Plasmodiumfalciparum, kills over a million children each year in Africa alone.

There is presently no effective vaccine against the parasite, and olderestablished drugs like chloroquine are rapidly loosing theireffectiveness due to resistance. On the other hand, ongoing research hasprovided many new antigens as potential malaria vaccine candidates.Several antibody targets on the malaria parasite have been identified,one of which is the circumsporozoite (CS) protein present on the surfaceof early sporozoites (Potocnjak et al., J. Exp. Med., 1980, 151:1504-1513). The central portion of the CS protein (M₁=⁴⁴ kDa contains 41tandem repeats of a tetrapeptide, 37 of which are Asn-Ala-Asn-Pro andfour of which are Asn-Val-Asp-Pro (Godson et al., Nature, 1983, 305:29-33; Dame et al., Science, 1984, 225: 593-599).

It was shown that linear tandemly repeated NANP peptides can elicitantibodies in mice and rabbits that recognize the native CS protein andblock sporozoite invasion of hepatocytes (Dame et al., Science, 1984,225: 593-599; Ballou et al., Science, 1985, 228: 996-999; Young et al.,Science, 1985, 228: 958-962; and Zavala et al., Science, 1985, 228:1436-1440). These results were a prelude to vaccination studies inhumans with synthetic (Asn-Ala-Asn-Pro)₃ peptides conjugated to tetanustoxin. However, the immune response was not strong enough for theseconjugates to be useful as malaria vaccine (Herrington et al., Nature,1987, 328: 257). Subsequently, a number of studies were initiated tooptimise the immune response to (Asn-Ala-Asn-Pro)_(n) peptides (Etlingeret al., Eur. J. Immunol., 1991, 21: 1505-1511; Tam et al., J. Exp. Med.,1990, 171: 299-306; Pessi et al., Eur. J. Immunol., 1991, 21: 2273-2276;and deOliviera et al., Vaccine, 1994, 12: 1012-1017).

All of these efforts should be seen in view of the fact that theconformations of the (Asn-Ala-Asn-Pro) repeats in the CS protein werenot known and so could not be taken into account in the design process.It seemed most likely that short linear (Asn-Ala-Asn-Pro)₃ peptideswould be largely unstructured in aqueous solution, and be susceptible torapid proteolytic degradation in serum. A later study also suggestedthat a significant part of the immune response against a linear(Asn-Ala-Asn-Pro)₃ peptide is directed against the chain termini, whichof course are not present in the native CS protein (Etlinger & Trzecjak,Phil. Trans. Roy. Soc. Lond. B. 1993, 340: 69-72). Although thestructure of the (Asn-Ala-Asn-Pro)-repeat region in the CS protein isstill unknown, modelling suggests that the (Asn-Pro Asn-Ala-Asn)-motifmay adopt a helical beta-turn, which is tandemly repeated in the CSprotein to generate a novel supersecondary structure. Cyclicpeptidomimetics of this (Asn-Pro Asn-Ala-Asn)-motif were synthesized andshown by NMR to adopt helical turns in aqueous solution (Pfeifer et al.,Chimia, 2001, 55(4): 334-339).

Using the approach to incorporate conformational constraints by means ofinternal cross-linking, U.S. Ser. No. 10/114,918 describes thepreparation of a conformationally constrained peptidomimeticcorresponding to the afore-mentioned tetrapeptide repeat region of theCS protein. In a five-fold repeat, one proline residue has been replacedby (2S,3R)-3-aminoproline and further, one alanine residue has beenreplaced by glutamate. Internal cross-linking has then been achieved byamide coupling between the primary amino group of the modified prolineunit and the side chain carboxyl group of glutamate.

As outlined above, peptide and protein mimetics are potentially of greatvalue in synthetic vaccine design. The mimetics should function bystimulating the immune system to produce antibodies that recognize theintact parasite. However, the difficulty of presenting in a mimetic theconformational epitopes found on the native antigenic that are requiredfor protective antibody responses are not yet fully overcome.

There is still a need for new routes to incorporate conformationalconstraints into a linear peptide chain to freeze a specificconformation. Since it is impossible to exactly predict the finalconformation of the resulting peptide, it is generally required tocreate a whole set of constrained peptides of the same primary sequence,which are then analysed for their ability to mimic an epitope of theantigenic protein. For example, such an analysis can be carried out bytesting the individual constrained peptides for their cross-reactivitytowards known antibodies of said antigenic protein. It is thereforenecessary to have a variety of different modes for incorporating aconformational constraint into a precursor peptide at hand.

Surprisingly, it has now been found that conformationally constrainedpeptides which are cyclized through a 4-substituted proline residue,having a specific stereochemistry, provide for a distinct and uniquethree-dimensional structure of a given peptide. The conformationalconstraint is achieved by an internal cross-link between a modifiedproline residue carrying a functional group at the 4-position and aspatially adjacent side chain functional group of a second residue. Themodified proline residue is characterised by a (2S,4S) stereochemistry.

Cyclic peptidomimetics cyclized via (2S,4S)-4-substituted prolineresidues provide for a new class of conformationally constrainedpeptides which appear to be more stable in aqueous solution andconsequently less prone to degradation in serum when, for example,administered as vaccine.

Synthesis of peptidomimetics containing (2S,4S)-4-substituted prolineresidue(s) are much easier accessible as for example their 3-substitutedcounterparts due to a shortened synthesis route comprising lessindividual steps. Furthermore, starting materials to synthesize the(2S,4S)-4-substituted proline unit are available in large amounts asthey are standard substances which can directly be purchased fromseveral suppliers, for example from Neosystem, 7 rue de Boulogne, 67100Strasbourg, France.

The incorporation of a (2S,4S)-4-substituted proline residue instead of,for example a 3-substituted proline unit achieves a conformationallyconstrained peptide which has a substantially altered three-dimensionalpeptide structure. It is surprising that this minor change of thechemical structure has such a strong impact on the conformation andimmunogenicity of the resulting peptide of the peptide.

Amino acids and amino acid residues described herein may be referred toaccording to the accepted one or three letter code referenced in textbooks well known to those of skill in the art, such as Stryer,Biochemistry, 4^(th) Ed., Freeman and Co., New York, 1995 and Creighton,Proteins, 2nd Ed. Freeman and Co. New York, 1993.

As used herein, the terms “peptide” and “polypeptide” are usedsynonymously and in their broadest sense to refer to a compound of twoor more amino acid residues, or amino acid analogs. The amino acidresidues may be linked by peptide bonds, or alternatively by otherbonds, e.g. ester, ether etc. As used herein, the term “amino acid” or“amino acid residue” refers to either natural and/or unnatural orsynthetic amino acids, including both the D or L enantiomeric forms, andamino acid analogs.

The term “epitope” or “B cell epitope” as used herein, designates thestructural component of a molecule that is responsible for specificinteractions with corresponding antibody (immunoglobulin) moleculeselicited by the same or related antigen. More generally, the term refersto a peptide having the same or similar immunoreactive properties, suchas specific antibody binding affinity, as the antigenic protein orpeptide used to generate the antibody. An epitope that is formed by aspecific peptide sequence generally refers to any peptide which isreactive with antibodies against the specific sequence.

The term “antigen” as used herein, means a molecule which is used toinduce production of antibodies. The term is alternatively used todenote a molecule which is reactive with a specific antibody.

The term “immunogen” as used herein, describes an entity that inducesantibody production in a host animal. In some instances the antigen andthe immunogen are the same entity, while in other instances the twoentities are different.

The term “immunopotentiating” is used herein to refer to an enhancingeffect on immune functions which may occur through stimulation of immuneeffector cells and may lead to increased resistance to infectious orparasitic agents.

The term “synthetic” as used herein relates to peptides produced by achemical method as described above, for example.

The term “peptidomimetic” is used herein to denote a peptide or peptideanalog that biologically mimics active determinants on parasites,viruses, or other bio-molecules.

The term “conformation” as used herein denotes the variousnonsuperimposable three-dimensional arrangements of atoms that areinterconvertible without breaking covalent bonds.

In a first embodiment, the present invention relates to a method forsynthesizing a conformationally constrained peptide which comprises oneor more regions of general formula (I):

(N-terminus) . . . -[X]_(m)-Y-[X]_(n)-Z-[X]_(o)- . . . (C-terminus)  (I)

wherein X=an amino acid residue or an amino acid analog and can be thesame of different if n>1;

m,o≧0 and n≧1, preferably ≧2;

Y,Z=A or B and Y≠Z, wherein

A=a 4-FgA-proline residue, wherein FgA is a functional group at the4-position of the proline residue;

B=an amino acid residue or an amino acid analog having a side chainfunctional group FgB,

wherein the functional groups FgA and FgB are capable of forming aninternal link by coupling the functional group FgA at the 4-position ofA and the side chain functional group FgB of B;

and comprises the following steps of (a) providing amino acid residues Yand Z having appropriate functional groups FgA and FgB, said functionalgroups being optionally protected, (b) synthesizing a linear peptidecomprising the amino acid residues Y and Z, and (c) optionallydeprotecting, if said functional groups are optionally protected, andreacting the functional groups FgA and FgB for converting the linearpeptide into the cross-linked form by coupling the functional group FgAat the 4-position of the proline residue (A) and the side chainfunctional group FgB of B.

Preferably, A is a (2S,4S)-4-FgA-proline residue, more preferably a(2S,4S)-4-aminoproline residue. Preferably, B is an amino acid residueor an amino acid analog having a side chain carboxyl group, preferablyglutamate or aspartate, wherein an internal cross-link is formed betweenA and B by amide coupling the amino group at the 4-position of the(2S,4S)-4-aminoproline residue and the carboxyl group of B.

Preferably, said conformationally constrained peptide is capable ofeliciting a pathogen-specific immune response in a mammal.

It is preferred that the linear peptide of step (b) comprises one ormore portions of the malaria circumsporozoite (CS) protein of aPlasmodium species, preferably Plasmodium falciparum. More preferably,this sequence comprises one or more tetrapeptides, which are selectedfrom the group consisting of Asn-Pro-Asn-Ala, Asn-Pro-Asn-Val,Asp-Pro-Asn-Ala and Asp-Pro-Asn-Val.

Preferably, the method for synthesizing conformationally constrainedpeptides according to the present invention is carried out using solidphase synthesis techniques in the assembling step (c). The linearpeptide can be assembled using Fmoc-chemistry. Cleavage from the resinand removal of side-chain protecting groups can proceed in one step andthe introduction of the internal cross-link between the(2S,4S)-4-substituted proline unit, preferably (2S,4S)-4-aminoproline,and a spatially adjacent side chain functional group can be achieved bycyclization in dimethylformamide (DMF) with a coupling reagent such asO-(7-Azabenzotriazole-1-yl)-N,N,N′,N-tetramethyluronium-hexafluorophospha-te(HATU).

Optionally, this method further includes the step of attaching thecyclized peptide to a phospholipid moiety (e.g. PE). The phospholipidanchor is preferably attached via a linker, preferably a dicarboxylatelinker, more preferably a succinate linker.

In another embodiment, this invention relates to conformationallyconstrained peptides which are obtainable by the method(s) according tothe invention. These peptides comprise one or more regions of generalformula (I):

(N-terminus) . . . -[X]_(m)Y-[X]_(n)-Z-[X]- . . . (C-terminus)  (I)

wherein X=an amino acid residue or an amino acid analog and can be thesame of different if n>1;

m,o≧O and n≅1, preferably ≧2;

Y,Z=A or B and Y≠Z, wherein

A=a 4-FgA-proline residue, wherein FgA is a functional group at the4-position of the proline residue;

B=an amino acid residue or an amino acid analog having a side chainfunctional group FgB,

wherein the functional groups FgA and FgB are forming an internal linkby coupling the functional group FgA at the 4-position of the prolineresidue and the side chain functional group FgB of B.

Preferably, A is a (2S,4S)-4-FgA-proline residue, more preferably a(2S,4S)-4-aminoproline residue.

Preferably, the functional groups FgA and FgB are chosen to form acyclic ester or cyclic amide bond. Preferably, B is an amino acidresidue or an amino acid analog having a side chain carboxyl group,preferably glutamate or aspartate, wherein an internal cross-link isformed between A and B by amide coupling the amino group at the4-position of the (2S,4S)-4-aminoproline residue and the carboxyl groupof B.

More preferably, the functional group at the 4-position of the modifiedproline unit is an amino group and cyclization is achieved through amidecoupling of said 4-amino of (2S,4S)-4-aminoproline (Apro) to a sidechain carboxyl group FgB of an spatially adjacent residue.

More preferred are peptides wherein the internal cross-link formingresidues A and B are separated by more than one amino acid residues oranalogs, i.e. n≧2.

Another embodiment of the invention relates to peptides which areconformationally constrained due to an internal cross-link between a(2S,4S)-4-functionalized proline unit and a suitable side chainfunctional group of a second residue and which are mimicking thethree-dimensional structure of, for example, an epitope on the surfaceof an antigenic protein. These peptides, designated hereinafter aspeptidomimetics, are characterized in that the above-described internalcross-link stabilizes a supersecondary 3D structure of the peptide.

To form this internal cross-link between the modified proline unit andthe side chain of another spatially adjacent amino acid residue oranalog, said side chain must provide a suitable functional group toallow the formation of a stable chemical bond between both residues.Preferably, the amino acid residue or analog B is selected from thegroup consisting of glutamate and aspartate and the afore-mentionedinternal cross-link is formed through amide coupling with(2S,4S)-4-aminoproline. More preferably, B is glutamate.

Therefore, the present invention also refers to the use of a4-Fg-proline, preferably a (2S,4S)-4-Fg-proline for synthesizingconformationally constrained peptides, wherein Fg is a functional group,selected from amino, hydroxy, sulfhydryl, halogen, sulfonyl, carboxy,thiocarboxy or substituted derivatives thereof, and being preferablyselected from amino, hydroxy, halogen, carboxy, or substitutedderivatives thereof. The invention furthermore refers to suchconformationally constrained peptides in which at least one4-Fg-proline, preferably a (2S,4S)-4-Fg-proline is incorporated.

The conformationally constrained peptides according to the presentinvention closely resemble the three-dimensional conformations found onan intact pathogenic protein, thus providing improved epitopes for thegeneration of pathogen-specific antibodies that efficiently cross-reactwith pathogens.

In yet another embodiment, the inventive peptide comprises one or moreportions of the malaria circumsporozoite (CS) protein of a Plasmodiumspecies, preferably of Plasmodium falciparum.

Protection of mammals, including man, against infection by the etiologicagent of malaria, Plasmodium can be achieved by eliciting an immuneresponse directed against the circumsporozoite (CS) protein. Fourspecies of Plasmodium are known to infect man. These are P. falciparum,P. vivax, P. ovale and P. malariae. The CS protein of P. falciparumcomprises about 412 amino acids with an approximate molecular weight of44,000. It comprises 41 tandem repeats of a tetrapeptide. Syntheticpeptides of a length of 5-20 residues derived from the repeat region ofthe CS protein of P. falciparum are preferred.

Although, the three-dimensional structure of the tetrapeptide repeatregion in the CS protein is still unknown, theoretical studies suggestthat it is likely to adopt a stable and repetitious conformation,possibly based on beta-helical turns or similar structures. The presentinvention provides peptides for the molecular mimicry of theconformational epitopes of the native malaria CS protein which arestructurally optimized in order to elicit cross-reactive antibodies withhigher efficiency.

In a preferred embodiment of the invention, the peptide comprises one ormore tetrapeptides selected from the group consisting ofAsn-Pro-Asn-Ala, Asn-Pro-Asn-Val, Asp-Pro-Asn-Ala and Asp-Pro-Asn-Val.More preferably, these peptides comprise 3 to 10, and most preferably 4to 6 of such tetrapeptide units.

The present invention relates to peptidomimetics for the, molecularmimicry of the conformational epitopes of the CS protein of Plasmodiumspecies, preferably P. falciparum. More preferably, the conformationallyconstrained peptide comprises (Asn-Pro-Asn-Ala), wherein n is 2, 3, 4 or5.

Models of these conformationally constrained peptidomimetics may beassessed for the stability and adoption of supersecondary structure inmolecular dynamics (MD) simulations in solvent. Adoption of asupersecondary structure by these model peptidomimetics may be evidencethat their structures are close to the preferred conformation of thetetrapeptide-repeat region in the native CS protein. Furthermore, 2DNOESY (nuclear Overhauser enhancement spectroscopy) spectra ofconformationally constrained peptidomimetics may be examined forconnectivities between the peptide NH groups in the tandemly repeatedhelical turns. Such connectivities provide evidence for the relativelystable helical turn formation in the context of a supersecondarystructure conformation.

In a more preferred embodiment, the invention relates to peptideswherein at least one of said Asn-Pro-Asn-Ala tetrapeptides is replacedby Asn-Pro-Asn-Glu and wherein the glutamate residue of Asn-Pro-Asn-Gluforms an internal cross-link through amide coupling with (2S,4S)-4-aminoproline.

Preferably, the peptide comprises one or more units of formula (II):

wherein Apro is (2S,4S)-4-aminoproline. It is more preferred that thepeptide essentially comprises a five-fold tandem repeat of(Asn-Pro-Asn-Ala) of the CS protein of P. falciparum, wherein Pro of thesecond repeat is replaced by a (2S,4S)-4-aminoproline unit and alanineof the fourth repeat is replaced by glutamate (formula III, hereinafterdenoted as UK39).

wherein Apro is (2S,4S)-4-aminoproline.

Through amide coupling, an internal cross-link is formed whichstabilizes a structure mimicking the native conformation of CS-proteintandem repeat epitopes.

As described in more detail in the following example, modeling studiesconcerning the peptidomimetic UK39 (formula V) have revealed a uniqueand distinct conformation compared to the isomeric mimetic BP66 whichmerely differs to UK39 in that the cyclization has been achieved through(2S,3R)-3-aminoproline. Due to the easily accessible(2S,4S)-4-aminoproline, UK39 (in contrast to BP66) can be obtained by ashort and standardized synthesis route. Since UK39 requires lessindividual synthesis steps and the starting material are catalogedstandard substances and are therefore available in large quantities,high amounts of UK39 can easily be obtained in higher amounts ifcompared to the corresponding synthesis of BP66.

Furthermore, UK39 is less prone to be degraded in aqueous solution thanits isomeric counterpart BP66. This is apparently due to a gain ofstability of the conformation. Moreover, UK39 shows an excellentantigenicity and parasite clearance in vivo. If compared with BP66, itis a better mimetic. With regard to cross reactivity to antibodies whichwere raised against P. falciparum, more antibodies bind to UK39 than toBP66.

The skilled artisan will appreciate that the afore-mentioned beneficialfeatures are not limited to amide bond cyclizations. That is to say,UK39 derivatives wherein the internal cross-link is formed by adisulfide bridge or by an ester bond, i.e. wherein the modified prolineunit is (2S,4S)-4-mercaptoproline or (2S,4S)-4-hydroxyproline,respectively, and wherein in the first case alanine is replaced bycysteine instead of glutamate are also encompassed by the presentinvention. The specific conformation behind the afore-mentionedbeneficial features is essentially determined by the stereochemistry andthe position of the cross-linking functional group at the modifiedproline ring.

The skilled artisan will appreciate that the afore-mentioned antigenicpolypeptide molecules may be administered with one or more adjuvants inorder to enhance the immunological response. For example, depending onthe host species, adjuvants which may be used include, but are notlimited to: mineral salts or mineral gels such as aluminum hydroxide,aluminum phosphate, and calcium phosphate; surface active substancessuch as lysolecithin, pluronic polyols, polyanions, peptides, oilemulsions, keyhole limpet hemocyanins, and dinitrophenol;immunostimulatory molecules, such as cytokines, saponins, muramyldipeptides and tripeptide derivatives, CpG dinucleotides, CpGoligonucleotides, monophosphoryl Lipid A, and polyphosphazenes;particulate and microparticulate adjuvant, such as emulsions, liposomes,virosomes, cochleates; or an immune stimulating complex mucosaladjuvants, Freund's (complete and incomplete, and potentially usefulhuman adjuvants such as BCG (bacille Calmette-Guerin) andcorynebacterium parvum.)

In another embodiment, the peptides according to the invention arecoupled to a phospholipid. Preferably, the N-terminus of these peptidesis coupled via a linker, preferably a dicarboxylate linker, morepreferably a succinate linker to a fatty acid derivative ofphosphaditylethanolamine, preferably1-palmitoyl-3-oleoyl-phosphatidylethanolamine (PE, formula IV). Saidfatty acid derivative is preferably a mono- to di-ester of glycerol withone or two C₁₀ to C₃₀ fatty acids which may have one or more doublebonds and may be same or different.

The peptides according to the invention can be used in synthetic vaccinedesign. Peptidomimetics function by stimulating the immune system toproduce antibodies that recognize the intact parasite. Preferably, suchmimetics are presented to the immune system in a way that leads to amore efficient antibody production. For example, cyclic peptidomimeticscan be presented on immunopotentiating reconstituted influenza virosomes(IRIVs) or liposomes, a form of antigen delivery that is practisedalready in human clinical use.

The attachment of a phospholipid anchor to the N-terminus ofpeptidomimetics functioning as antigen allows to combine the peptidewith immunopotentiating delivery systems. According to a further aspectof the invention, peptides are thus preferred which are combined with animmunopotentiating delivery system. Preferred immunopotentiatingdelivery systems are selected from the group consisting of liposomes,multiple-antigen peptides and immunopotentiating reconstitutedvirosomes.

For example, immunopotentiating reconstituted influenza virosomes (IRIV)as human compatible immunopotentiating delivery agents are capable ofpresenting the conformationally constrained peptidomimetics in multiplecopies to the immune system and therefore, further improves thegeneration of efficient pathogen cross-reactive antibody responses.IRIVs are spherical, unilamellar vesicles, prepared from a mixture ofphospholipids and influenza virus surface glycoproteins. Thehemgglutinin membrane glycoprotein of influenza virus plays a key rolein the mode of action of IRIVs. This major antigen of influenza is afusion-inducing component, which facilitates antigen delivery toimmunocompetent cells. In addition, peptides according to the invention(T cell epitopes) can be encapsulated into virosomes in order to beprotected from enzymatic degradation by the body fluids and will bepresented to the immune system via the MHC I class pathway.

Particularly preferred is a peptide of five (Asn-Pro-Asn-Ala) tandemrepeats that is internally cross-linked as described above and whichfurther comprises a PE moiety at the N-terminus which provides for theattachment to an immunopotentiating reconstituted influenza virosome(formula V).

For in vivo experiments peptides according to the invention can becombined with adjuvants in order to enhance the immunological response.For example, depending on the host species, adjuvants which may be usedinclude, but are not limited to: mineral salts or mineral gels such asaluminum hydroxide, aluminum phosphate, and calcium phosphate; surfaceactive substances such as lysolecithin, pluronic polyols, polyanions,peptides, oil emulsions, keyhole limpet hemocyanins, and dinitrophenol;immunostimulatory molecules, such as cytokines, saponins, muramyldipeptides and tripeptide derivatives, CpG dinucleotides, CpGoligonucleotides, monophosphoryl Lipid A, and polyphosphazenes;particulate and microparticulate adjuvant, such as emulsions, liposomes,virosomes, cochleates; or an immune stimulating complex mucosaladjuvants, Freund's (complete and incomplete, and potentially usefulhuman adjuvants such as BCG (bacille Calmette-Guerin) andcorynebacterium parvum.) However, it is noted that strong adjuvants,e.g. Freund's adjuvants can cause severe undesirable side effects thatthey are not accepted by regulatory authorities for human use.

Yet another embodiment concerns the use of (2S,4S)-4-substituted prolinefor synthesizing conformationally constrained peptides. Preferably, thiscovalently modified proline is used in one of the above described methodfor synthesizing conformationally constrained peptides which have beencyclized through coupling of said modified proline unit, preferably(2S,4S)-4-aminoproline, and a second residue of the peptide chain. Inthis method, (2S,4S)-4-aminoproline is incorporated during theassembling step (b).

The cross-linked peptidomimetic can be prepared by solid phase synthesismethods well-known in the art. The linear peptide can be assembled usingFmoc-chemistry. Cleavage from the resin and removal of side-chainprotecting groups can proceed in one step and the key backbone couplingof the modified proline residue and glutamate can be achieved bycyclization in DMF with a coupling reagent such as HATU.

In a further embodiment, the invention relates to the use of theinventive peptides for the manufacture of a vaccine for the treatment ofmalaria. Preferably, conformationally constrained peptidomimetics of thecentral (Asn-Pro-Asn-Ala) repeat region of the CS protein of the malariaparasite P. falciparum can be used to mimic the surface structure of CSprotein and thereby elicit a humoral immune response. Antibodies raisedagainst such a mimetic are capable to cross-react with the native CSprotein on P. falciparum sporozoites. Therefore, peptidomimeticsaccording to the invention can be widely used in the design ofmolecularly defined combined synthetic vaccines, including thosetargeted against multiple antigens and development stages of P.falciparum, and against other infectious agents.

In a further embodiment, the invention concerns a method for producingantibodies in a host against Plasmodium species, preferably againstPlasmodium falciparum comprising the step of administering anabove-described peptide to said host.

Another embodiment of the present invention relates to an in vitromethod for detecting Plasmodium species in a sample comprising the steps(a) contacting said sample with an antibody according to the inventionunder conditions such that binding to CS protein epitopes occurs if CSprotein is present; and (b) detecting the presence of said antibodybound to an CS protein epitope.

In detail, the method comprises incubating a test sample with one ormore antibodies of the present invention and assaying whether theantibody binds to the test sample. The presence of CS protein mayindicate malaria disease.

Conditions for incubating an antibody with a test sample vary.Incubation conditions depend on the format employed in the assay, thedetection methods employed and the type and nature of the antibody usedin the art. Examples of such assays can be found in Tijssen, “Practiceand theory of enzyme immunoassays: Laboratory Techniques in Biochemistryand Molecular Biology,” Elsevier Science Publishers, NL (1985).

Yet another embodiment of the present invention relates to a kit fordetecting the presence of Plasmodium species in a sample, wherein saidkit comprises: (i) a first container means containing an antibodyaccording to the invention, and (ii) second container means containing aconjugate comprising a binding partner of the antibody and a label. Inanother preferred embodiment, the kit further comprises one or moreother containers comprising one or more of the following: wash reagentsand reagents capable of detecting the presence of bound antibodies.

Examples of detection reagents include, but are not limited to, labeledsecondary antibodies, or in the alternative, if the primary antibody islabeled, the chromophoric, enzymatic, or antibody binding reagents whichare capable of reacting with the labeled antibody.

Having now generally described the present invention, the same may bemore readily understood by reference to the following example inconnecting with the accompanying FIGS. 1-3.

FIG. 1 schematically illustrates the route of synthesis to theconformationally constrained peptidomimeticAsn-Pro-Asn-Ala-Asn-(Apro-Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Glu)cyclo-Asn-Pro-Asn-Alaattached to a PE moiety.

FIG. 2 shows the grade of purification of the inventive peptidomimeticaccording to formula (IV) in an HPLC chromatogram. After completing thesynthesis, the solvent was removed and, the resulting residue purifiedusing a C4 reverse phase HPLC column (Vydac 214 TP 1010, 25 cm.times.10mm) using a gradient starting with 50% ethanol in water to 100% ethanol(+0.1% TFA) over 15 minutes. UK39 appears as a broad peak at about 90%ethanol. m/z 1427 (M+2H)²⁺.

FIG. 3 shows a comparison of average NMR structures in aqueous solutionof the prior art peptide BP66 and the peptide UK39 according to thepresent invention. BP66 only differs to UK39 in that the modifiedproline unit (2S,4S)-4-aminoproline has been replaced by the(2S,3R)-3-aminoproline isomer. Due to the change of the position of thecross-link from the 3- to the 4-position, the conformation of the entiremacrocyclic portion of the peptide has been changed. The figure showsaverage NMR structures deduced in water by NMR and dynamic simulatedannealing (SA). The figure was prepared using MOLMOL (Konradi R. et al.,J. Mol. Graph. 1996, 14, 51-55).

It should be understood that the following example is for illustrativepurposes only and should not be construed as limiting this invention inany way to the specific embodiment recited therein.

Unless otherwise specified, general chemical and peptide synthesisprocedures, such as those set forth in Voet, Biochemistry, Wiley, 1990;Stryer; Peptide Chemistry. A Practical Textbook, 2nd ed., MiklosBodanszky, Springer-Verlag, Berlin, 1993; Principles of 15 PeptideSynthesis, 2nd ed., Miklos Bodanszky, Springer-Verlag, Berlin, 1993;Chemical Approaches to the Synthesis of Peptides and Proteins, P.Lloyd-Williams, F. Albericio, E. Giralt, CRC Press, Boca Raton, 1997;Bioorganic Chemistry: Peptides and Proteins, S. M. Hecht, Ed., OxfordPress, Oxford, 1998, are used.

EXAMPLES Synthesis and Purification of the Inventive PeptidomimeticAccording to Formula (V)

The following demonstrates the synthesis of the conformationallyconstrained peptidomimeticAsn-Pro-Asn-Ala-Asn-(Apro-Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Glu)cyclo-Asn-Pro-Asn-Alaattached to a PE moiety. The synthesis of the linear peptide precursorwas performed on Rink Amide MBHA resin (0.73 mM/g) (Novabiochem) on anApplied Biosystems ABI433A peptide synthesizer. The peptide wassynthesized on a 0.25 mmol scale using 4 eq of each Fmoc-protected aminoacid each activated with HBTU/HOBt (4 eq.). The amino acids used were:Fmoc-Asn(Mtt)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH andFmoc-(4S,2S)-4-aminoproline(Boc)-OH.

The cleavage of the linear peptide from the resin was carried out usingTFA containing 2.5% TIS and 2.5% water over 3 h at room temp. Afterremoval of most of the TFA in vacuo, the peptide was precipitated usingdiisopropyl ether, washed with iPr₂O, and dried in vacuo for 1 h. Theproduct can be analyzed by reverse phase HPLC on a C18 column using agradient of MeCN/H₂O (+0.1% TFA; 5→95% MeCN; t_(R)=10 min). m/z: 2294(M+H)⁺.

For cyclization, the crude linear peptide from above was stirredovernight at room temp. together with 4 eq HATU, 4 eq HOAt in DMF and 1%v/v DIEA (2 mg/ml peptide). The solvent was removed and the peptidedissolved in 20% piperidine/DMF and stirred for 15 min. at room temp. toremove the Fmoc group. The solvent was evaporated and peptide wasprecipitated using diisopropyl ether and dried in vacuo. The product canbe analyzed by reverse phase HPLC on a C18 column using a gradient ofMeCN/H₂O (+0.1% TFA; 5→95% MeCN; t_(R)=11 min). m/z: 2276 (M+H)⁺.

The foregoing product (40 mg) in DMF (5 ml) with 4 eq each of HATU/HOAtwas treated with a solution of PE-CO—(CH₂)₂—COOH (PE-succinate; 4 eq.)in DCM (5 ml) together with 1% of DIEA and stirred overnight at roomtemp. The solvent was removed and the resulting residue purified using aC4 reverse phase HPLC column (Vydac 214 TP 1010, 25 cm.times.10 mm)using a gradient starting with 50% ethanol in water to 100% ethanol(+0.1% TFA) over 15 minutes. UK39 appears as a broad peak at about 90%ethanol. m/z 1427 (M+2H)²⁺ (see below).

Cross-Reactivity to Antibodies Raised Against the CS Protein of P.falciparum

The following demonstrates the cross-reactivity of both UK39 and theisomeric form BP66 to antibodies raised against the CS protein of P.falciparum. BP66 merely differs to UK39 in that the modified prolineunit (2S,4S)-4-aminoproline (formula VI) of UK39 has been replaced bythe (2S,3R)-3-aminoproline isomer.

Table 1 shows that more monoclonal antibodies raised against the CSprotein bind to UK39 than to BP66. Peptidomimetic UK39 comprising(2S,4S)-4-aminoproline thus provide for an improved mimicking of thetandem repeat region of the CS protein

TABLE 1 Immunological cross-reactivity of monoclonal antibodies raisedagainst P. falciparum sporozoites with the closely related mimotopesUK39 and BP66. Mab BP 66 UK 39 Sp4-5F2 + + Sp4-2H1 + + Sp3-E6 + +Sp3-C6 + + Sp3-E9 + + Sp4-4B6 + + Sp4-7C2 + + Sp4-7E4 + + Sp4-7H1 − +Sp4-4D7 + + Sp3-B4-C12 + + Sp4-1B4 − +

The benefits in immunological reactivity arise from the use of(2S,4S)-4-aminoproline rather than the earlier (2S,3R)-3-aminoproline.So the improvement arises by moving the site of cross-linking from the3- to the 4-position of the proline ring. The immunological responsegenerated by the novel mimetic is stronger and different. Thisdifference is seen in the fact that a monoclonal antibody could beisolated from the immunization with the mimetic containing(2S,4S)-4-aminoproline that binds the parasite but does not bind themimetic containing 3-aminoproline. This indicates that the new moleculemimics parts of the parasite surface protein that are not represented atall in the earlier mimetic containing (2S,3R)-3-aminoproline (theantibody binds the parasite and the mimetic with (2S,4S)-4-aminoprolinebut not the mimetic with 2S,3R)-3-aminoproline). This can be easilyrationalized, since by changing the position of the cross-link from the3-position to the 4-position the inventors inevitably changed theconformation (shape) of the macrocyclic portion of the molecule. This isa crucial part for recognition by antibodies. Molecular modellingstudies with both mimetics supports the notion that changing theposition of the cross-link also changes the conformation of thebackbone. The change in conformation may be small. But even smallchanges in conformation may lead to changes in the way the mimetic isrecognized by antibodies, and hence change the ability of the moleculeto mimic epitopes on the surface of the parasite (Table 1). Modelling ofthe Conformation of the Conformationally Constrained PeptidomimeticsUK39 and BP66

To determine which conformation the constrained peptide antigen willadopt in aqueous solution, the peptidomimetic can be studied by NMR andMD methods in aqueous solution in analogy to previous studies (Bisang,C. et al., J. Am. Chem. Soc. 1998, 120, 7439-7449). Average solutionstructures for the conformationally constrained peptidomimetics arecalculated using NOE-derived distance restraints by dynamic simulatedannealing (SA) and moleculare dynamics (MD.) simulations, using methodsdescribed earlier (Bisang, C. et al., J.

Am. Chem. Soc. 1998, 120, 7439-7449)

Modeling studies concerning UK39 and BP66 show that cyclization throughthe (2S,4S)-4-aminoproline residue stabilizes the conformation of UK39.Moreover, modeling reveals that BP66 adopts a different structure thanUK39. Therefore, it can be concluded that the change of the cyclizationfrom position 3 to 4 together with the specific stereochemistry of theprimary amino group at the modified proline residue creates a newconformation in the NPNA motifs.

Preparation of Mimetic-Loaded Virosomes

For the preparation of PE-mimetic-IRIV, a solution of 4 mg purifiedInfluenza A/Singapore hemagglutinin is centrifuged for 30 min at 100,000g and the pellet is dissolved in 1.33 ml. of PBS containing 100 mM OEG(PBS-OEG). 32 mg phosphatidylcholine (Lipoid, Ludwigshafen, Germany), 6mg phosphatidylethanolamine and the PE-mimetics are dissolved in a totalvolume of 2.66 ml PBS-OEG. The phospholipids and the hemagglutininsolution are mixed and sonicated for 1 min. This solution is thencentrifuged for 1 hour at 100,000 g and the supernatant is sterilefiltered (0.22 μm). Virosomes are then formed by detergent removal usingBioRad SM BioBeads (BioRad, Glattbrugg, Switzerland).

Immunogenicity Studies for the UK39-Peptidomimetic-IRIV

Antibody responses elicited by IRIVs loaded with the conformationallyconstrained peptidomimetic UK39 are studied in BALB/c mice.Preimmunization is achieved with the influenza vaccine Inflexal Berna™(Berna-Products, Bern, Switzerland). Immunization is achieved withseveral doses of IRIV-peptidomimetic.

BALB/c mice are preimmunized intramuscularly with commercial whole virusinfluenza vaccine (0.1 ml; Inflexal Berna, Berna Products, Bern,Switzerland) on day 21. Starting on day 0, they received at three-weeklyintervals three doses of conformationally constrained UK39peptidomimetic linked to IRIV intramuscularly at doses of 50 μg ofmimetic. Blood is collected two weeks after the third immunization andanalyzed by ELISA and IFA.

Enzyme-Linked Immunosorbent Assays (ELISA)

ELISA microtiter plates (Immunolon 413, Dynatech, Embrach, Switzerland)are coated at 4° C. overnight with 50 ml of a 5 mg/ml solution ofpeptidomimetic constructs in PBS, pH 7.2. Wells are then blocked with 5%milk powder in PBS for 1 h at 37° C. followed by three washings with PBScontaining 0.05% Tween-20. Plates are then incubated with twofold serialdilutions of mouse serum or hybridoma cell supernatants in PBScontaining 0.05% Tween-20 and 0.5% milk powder for 2 h at 37° C. Afterwashing, the plates are incubated with alkaline phosphatase-conjugatedgoat anti mouse IgG (g-chain specific) antibodies (Sigma, St. Louis,Mo.) for 1 h at 37° C. and then washed. Phosphatase substrate (1 mg/mlp-nitrophenyl phosphate, Sigma) in buffer (0.14% Na₂CO₃, 0.3% NaHCO₃,0.02% MgCl₂, pH 9.6) is added and incubated at room temperature. Theoptical density (OD) of the reaction product is recorded afterappropriate time at 405 nm using a microplate reader (Titertek MultiscanMCC/340, Labsystems, Finland). Titration curves are registered andanalyzed using GENESIS LITE 2.16 software (Life Sciences Ltd.,Basingstoke, UK). Effective dose 20% values (ED20%) are calculated foreach curve and the corresponding titers are set as endpoint titers.

Immunofluorescence Assays

Immunofluorescence assays are performed to assess cross-reactivity ofthe antibodies obtained. Air-dried unfixed P. falciparum salivary glandsporozoites (strain NF54) attached to microscope glass slides areincubated in a moist chamber for 20 min at 37° C. with serum diluted inPBS. The slides are then washed five times with PBS containing 0.1%bovine serum albumin (PBS-BSA) and dried. FITC-labelled goat anti-mouseIgG (Fab-specific) antibodies (Sigma), optimally diluted in PBScontaining 0.1 μl Evans43lue (Merck, Germany), are added. Afterincubation for 20 min at 37° C. the slides are again washed five timeswith PBS-BSA, dried, mounted with glycerol, and covered with a coverslide. A Leitz Dialux 20 microscope using 12.5/18 ocular and a 40×/1.30oil fluorescence 160/0.17 objective is used to detect fluorescencestaining at 495 nm excitation and 525 nm emission wavelength.

1. A malaria vaccine comprising: a peptide comprising one or more unitsof formula (II):

wherein Apro is (2S,4S)-4-aminoproline; and one or more pharmaceuticallyacceptable adjuvants.
 2. The malaria vaccine of claim 1, wherein thepeptide comprises the formula (III):


3. The malaria vaccine of claim 1, wherein the peptide is coupled to aphospholipid.
 4. The malaria vaccine of claim 3, wherein the N-terminusof the peptide is coupled via a linker to a phosphatidylethanolamine. 5.The malaria vaccine of claim 4, wherein the linker is a dicarboxylatelinker.
 6. The malaria vaccine of claim 5, wherein the dicarboxylatelinker is a succinate linker.
 7. The malaria vaccine of claim 4, whereinthe phosphatidylethanolamine is1-palmityl-3-oleoylphosphatidylethanolamine (PE).
 8. The malaria vaccineof claim 1, wherein the peptide is combined with an immunopotentiatingdelivery system selected from the group consisting of liposomes,multiple-antigen peptides, and immunopotentiating reconstitutedvirosomes.
 9. The malaria vaccine of claim 2, further comprising a1-palmityl-3-oleoylphosphatidylethanolamine (PE) moiety at theN-terminus attached to an immunopotentiating reconstituted virosome. 10.The malaria vaccine of claim 1, wherein the peptide can mimic thesurface structure of a circumsporozoite (CS) protein, thereby elicitinga humoral immune response.
 11. The malaria vaccine of claim 1, whereinthe peptide can elicit the production of an antibody capable ofcross-reacting with a native circumsporozoite (CS) protein of aPlasmodium.
 12. A method of producing a malaria vaccine comprising:providing a peptide comprising one or more units of formula (II):

wherein Apro is (2S,4S)-4-aminoproline and providing one or morepharmaceutically acceptable adjuvants.
 13. The method of claim 12,wherein the peptide comprises the formula (III):


14. The method of claim 12, wherein the peptide is coupled to aphospholipid.
 15. The method of claim 14, wherein the N-terminus of thepeptide is coupled via a linker to a phosphatidylethanolamine.
 16. Themethod of claim 15, wherein the linker is a dicarboxylate linker. 17.The method of claim 16, wherein the dicarboxylate linker is a succinatelinker.
 18. The method of claim 15, wherein the phosphatidylethanolamineis 1-palmityl-3-oleoylphosphatidylethanolamine (PE).
 19. The method ofclaim 12, wherein the peptide is combined with an immunopotentiatingdelivery system selected from the group consisting of liposomes,multiple-antigen peptides, and immunopotentiating reconstitutedvirosomes.
 20. The method of claim 13, further comprising a1-palmityl-3-oleoylphosphatidylethanolamine (PE) moiety at theN-terminus attached to an immunopotentiating reconstituted virosome. 21.The method of claim 12, wherein the peptide can mimic the surfacestructure of a circumsporozoite (CS) protein, thereby eliciting ahumoral immune response.
 22. The method of claim 12, wherein the peptidecan elicit the production of an antibody capable of cross-reacting witha native circumsporozoite (CS) protein of a Plasmodium.